Mammalian nuclear transfer procedures were developed in the late 1980s. The basic nuclear transfer procedure includes the enucleation of an oocyte in metaphase II (MII), and the transfer of a donor nucleus by fusion or injection into the enucleated oocyte. An important aspect of nuclear transfer is the reprogramming of the donor nucleus. Nuclear reprogramming refers to modifying a nucleus so the nucleus is capable of directing development from the one-cell embryo stage to offspring.
The first cloned rabbit was produced using embryonic cells as a source of donor nuclei. Over the last decade, the source of the donor has been expanded to include differentiated cells as well (Table 1). For example, the first cloned transgenic calves were produced recently using donor nuclei from fetal cells. Both studies used unfertilized MII oocytes that were first enucleated and then fused with the donor cell.
TABLE 1Species and donor cell type usedto produce cloned mammals.Cell type used to produce a nucleartransfer offspring (clones)SpeciesEmbryonicFetalAdultMouseCheong et al., 1994None reportedWakayama et al.,1998RabbitStice and Robl,None reportedNone reported1988CattlePrather et al., 1987Cibelli et al., 1998Kato et al., 1998SheepWilladsen, 1986Campbell et al.,Wilmut et al., 19971996PigPrather et al., 1989Onishi et al., 2000Polejaeva et al.,2000Citations: Campbell et al., Nature, 380, 64 (1996); Cheong et al., Biol. Reprod., 48, 958 (1993); Cibelli et al., Science, 280, 1256 (1998); Kato et al., Science, 282, 2095 (1998); Onishi et al., Science, 289, 1188 (2000); Prather et al., Biol. Reprod., 37, 859 (1987); Polejaeva et al., Nature, 407, 86 (2000); Prather et al., Biol. Reprod.., 41, 414 (1989); Stice et al., Biol. Reprod., 39, 657(1988); Wakayama et al., Nature, 394, 369 (1998); Willadsen et al., Nature, 320, 63 (1986); and Wilmut et al., Nature, 385, 810 (1997).
Successful cloning using undifferentiated embryonic cells versus differentiated cells as a source of donor nuclei for introduction to an MII oocyte may depend on the order in which fusion and activation are performed. Bovine embryonic cell-derived clones developed at a higher rate when the MII oocyte was activated first followed by introduction of the donor nucleus into the activated oocyte (Barnes et al., Mol. Reprod. Dev., 36, 33 (1993); Stice et al., Mol. Reprod. Dev., 38, 61 (1994)). Bovine fetal and adult cell cloning was accomplished by reversing the fusion and activation steps in the cloning process, and resulted in the first cloned cattle fetuses from differentiated cell lines, and later in offspring from fetal cells (Cibelli et al., Science, 280, 1256 (1998); Stice et al., Biol. Reprod., 54, 100 (1996)).
In addition, the state of the donor cell used for cloning has varied. Dolly was the result of using donor cells that were quiescent (Wilmut et al., Nature, 385, 810 (1997)). However, other studies using quiescent cells have produced very different results. Various mouse cells that are naturally in a quiescent state (cumulus cells, sertoli cells and neural cells) were harvested and used in cloning procedures. The cumulus cells gave rise to offspring while the other quiescent cells did not. Arguably, the least quiescent of the three cell types is the cumulus cells since these are often mixed with granulosa cells which will propagate very well in culture. Cibelli and coworkers (Science, 280, 1256 (1998); Stice et al., (U.S. Pat. No. 5,945,577)) demonstrated that non-serum starved proliferating bovine fetal fibroblast cells were a suitable donor source for nuclear transfer with efficiencies similar to reports using serum-starved (i.e., quiescent) cells. In addition, adult mouse fibroblast cells cultured in serum and no serum were compared but both groups resulted in low developmental rates to term. To date no firm conclusion can be made on whether quiescent or proliferating cells are the best sources of donor cells for nuclear transfer. Neither methods using quiescent cells nor proliferating cells appear to result in marked improved cloning efficiencies or outcomes.
Improvements in oocyte activation in various species have been vigorously pursued (reviewed in Prather et al., Theriogen., 51, 498 (1999)). Progress has been made by increasing calcium and/or decreasing protein phosphorylation in the oocyte (mice, Szollosi et al., J. Cell Sci., 104, 861 (1994); cattle, Susko-Parrish et al., Dev. Biol., 166, 729 (1994) and Susko-Parrish et al., (U.S. Pat. No. 5,496,720)).
Cloning pigs in particular is technically difficult. A cloned pig derived from four-cell stage embryo nucleus was reported in 1989 (Prather et al., Biol. Reprod., 41, 414-8 (1989). Some groups have produced blastocyst stage pig nuclear transfer embryos derived from differentiated cells (Table 2).
TABLE 2Procine fetal fibroblast cells (G0/G1) fusedinto enucleated MII oocytes and developmentof resulting nuclear transfer (NT) embryos.Number of NTNumberembryos developingof NTto morulaembryosand blastocystReferenceproducedstage (%)Number of offspringDu et al., 1999815 (8)Quality too poor totransferMiyoshi et al.,361 (3)Quality too poor to1999transferTao et al., 1999100 3 (7)Average nuclei in blastwas 19.5Citations: Du et al., Theriogenology, 51, 201 (1999); Miyoshi et al., Theriogen., 51, 210 (1999); and Tao et al., Cloning, 1, 55 (1999).
Improvements in porcine oocyte activation have lagged behind other species, particularly in development of the activated unfertilized oocyte (i.e., parthenogenetic development). Recently, pig cloning has been reported (Onishi et al., Science, 289, 1188 (August, 2000); Polejaeva et al., Nature, 407, 86 (September, 2000); and Betthauser et al., Nature Biotechnol., 18, 1055 (October, 2000)). However, nuclear transfer embryo developmental rates with in vitro and in vivo derived MII oocytes remain poor. Therefore, there is a need to employ novel changes in the nuclear transfer procedure to produce cloned offspring from differentiated cell types.